Lipof/Opti transfection cells died in DMEM/F12 - Transfection (Apr/28/2005 )

I am transfecting some drug receptors in HEK 293 cells using Lipofectamine 2000 in Opti MEM from Invitrogen. The protocol said that I can replace the Opti MEM (without serum) with the regular medium after 24 hours. However, when I loaded the regular medium, the good-looking healthy cells just aggregate/fuse together within a few hours, and started to float away from the plate bottome eventually. Firstly, I think that might be due to the presence of antibiotic of the regular medium. But even after I took out the antibiotic, still those HEK cells more like to stay in the serum free Opti MEM after transfection. Even they grow so well in the regular medium with antibiotic before transfection.

Help me please and thanks!!!

Lipofectamine or lipofectamine2000 with longer exposure (>12 hr) caused annoying cytotoxicity on many cells - it's a well known fact!
Our lab routinely used GenCarrier-1 reagent from Epoch Biolabs on C6 glioma, PC-12, HEK293, COS and 3T3 cells and get very reproducible results each time.
That's why we finally switch to GenCarrier which is much less toxic while still maintain high Tfx efficiency. In addition, GenCarrier-1 is much cheaper.

hi
usually during transfection, it's necessary not to use serum and not to use antibiotic.
For 293cells, 3hours after transfection you have two alternatives :
change the medium for fresh one
let the transfection medium over the cells but "complete" it with a 2xserum containing medium (without antibiotics)

i've heard once that lipofectamine 2000 was less toxic than lipofertamine


Hope that helps...
fred

How confluent are your cells? 293s are famous for lifting off the plate with only the slightest amount of disruption especially if they are quite confluent.

Thanks a lot!!

The cells are around 70 to 90%. They are very good looking guys when they are in the culture medium (DMEM/F12,10% serum and GluMax) without transfection.

Now I did sth and the cells are better looking now.

First, I replaced the cells with OptiMEM after five hours transfection. Then add 1/2 part (10ml OptiMEM for 5ml ) culture medium after 24 hours transfection and then replace them with the culture medium after 48 hours transfection. I dont know the rationale, but the cells are good now. The point is, I found the transfectected HEK 293T cells like the OptiMEM very much and I gradually weaned them from the OptiMEM and gave them regular serum contained medium. I guess the cells might be very fragile after transfection and couldnt tolerate any harsh changes, like pH, since regualr medium needs CO2 buffered pH (take a while) and OptiMEM has HEPES to buffer pH right away.